SUMMARY ? Project 2 The broad, long-term goal of our research program is to advance knowledge of viral replication strategies and virus?host cell interactions that are relevant for therapeutic intervention. The viruses that we study are positive- strand RNA viruses including dengue virus (DENV), hepatitis C virus (HCV), and Zika virus (ZIKV). To complement animal models, there is a clear need for better in vitro models to study infectious diseases that affect largely inaccessible organs such as the human nervous system. In the current HTMID CRC proposal with the overarching goal to develop an organoid-based platform using human induced pluripotent stem cells (iPSCs), Project 2 focuses on ZIKV, which poses a major emerging threat to human health due to a large number of recent outbreaks and its association with microcephaly, a neurodevelopmental birth defect. Understanding how ZIKV interacts with the host to cause disease is of critical importance to the development of effective antiviral drugs and a prophylactic vaccine, both of which are urgently needed. We have recently published work showing that ZIKV infects human neural stem cells and also performed a high-throughput screening to identify small molecule compounds that can inhibit ZIKV infection. Here we propose to utilize a 3D cerebral organoid model to investigate the mechanism of ZIKV pathogenesis in human neural development (Specific Aims 1 & 2). We will also use the organoid model to compare and contrast ZIKV with West Nile virus (WNV), another human neurotropic virus of biomedical importance (Specific Aim 3). Finally, we will validate the utility of the organoid model as a drug testing tool using chemical compounds with confirmed antiviral or anti-apoptotic functions (Specific Aim 4). With the successful completion of the project, we expect to: positively identify the type of cells and their ZIKV infection rate during the different stages of brain development; unravel the mechanisms by which ZIKV infection causes neuronal death and developmental defects; reveal any quantitative differences between ZIKV and WNV in infection efficiencies, cellular tropism, and pathology in the 3D model; and confirm lead compounds for inhibiting ZIKV replication in both 2D culture and 3D tissue models. These results will validate the utility of the 3D brain organoid model for both basic and translational research of ZIKV and also provide direct and immediate impact on the mission to develop effective therapy to treat ZIKV infection and its associated neurological diseases.